(B) Western blots were used to test for NleA levels in the bacterial pellets
(P) or in the medium [secreted (S)] produced by wild-type EPEC (wt) or
mutants (DsepL, DsepD, DnleA, DescV, DescN, and Dces T) grown in Dulbecco’s
modified Eagle’s medium (DMEM). Some mutants were supplemented
with plasmids expressing the wild-type alleles of sepL and sepD (pSepL and
pSepD, respectively). (C) EPEC (wt), EPECnleA-gfp, and a Dces T mutant,
with or without a plasmid (pCes T), were grown in DMEM. IPTG was added
to activate Ces T expression. Western blots and antibodies to NleA and
Ces T (anti-NleA and anti-Ces T, respectively) were used to test for levels of
NleA, NleA-GFP, and Ces T in the bacteria. (D) E. coli MC1061 (wt) or a DcsrA

mutant containing pnleA-gfp or both pnleA-gfp and pCes Tplasmids were
gro wn in DMEM, with or without the addition of IP TG (to induce Ces Tproduc-tion). Western blots and antibodies to GFP were used to test for the levels of
NleA-GFP in the bacteria. (E) Lysates containing CsrA-SBP, GST, or GST-Ces T were mixed as indicated and pulled down by glutathione agarose
beads. The inputs and captured proteins were detected by Western blots
using anti-GSTor anti-SBP. IP, immunoprecipitation. (F) Ces Tantagonizes
binding of CsrA to the nleA 5′UTR. Labeled #3 RNA [0.6 nM (fig. S7)] was
incubated with or without CsrA (5 mM) and with or without increasing concentrations of GST-Ces T (5 to 20 mM) or GST (negative control, 25 mM) and
subjected to gel mobility shift assays.

Fig. 2. CsrA and the nleA 5′UTR act together to repress NleA expression.

(A) Plasmids containing nleA and its regulatory region fused to gfp are shown.
These include transcription (a) or translation (b) fusions, replacement of the
nleA promoter with the tet promoter (c and d), deletion of the nleA ORF (e),
replacement of the native 5′UTR with a synthetic one (f), and internal deletion
within the 5′UTR (g). Black and green lines represent the nleA and tet regulatory
regions, respectively, and the arrows indicate promoters. Red and blue lines
represent the nleA and synthetic 5′UTR, respectively. Plasmid names (and numbers) are indicated. (B to E) Bacteria cultures grown under infection conditions were extracted, and GFP expression levels were analyzed by Western blots.

The tested bacteria included EPEC, E. coli K12 (strain MC1061), and their re-spective DcsrA mutants containing the plasmids described in (A), as indicatedabove the lanes (a to g). Locations of GFP and NleA-GFP are denoted by arrowsand NleA-GFP degradation products [(C) to (E)] by arrowheads. In (C), Tet wasadded to activate the tet promoter. (E) wt, wild type. (F) NleA expression by theEPEC DcsrA mutant with or without a plasmid expressing CsrA via the isopropyl-b-D-thiogalactopyranoside (IPTG)–regulated promoter (pCsrA-SBP). The pres-ence of the plasmid and IPTG concentrations are indicated. (G) Binding of CsrAto the nleA 5′UTR. Labeled RNA (0.6 nM) were incubated with serial 1:2 dilutionsof CsrA (starting with 12 mM) and tested by gel mobility shift assays. The testedRNAs include a negative control (#4 RNA), putative CsrA binding sites in thenleA 5′UTR (#1 and #3 RNA), and a positive control (PC RNA) (figs. S5 and S7).RESEARCH | REPORTS